John M. S. Bartlett.pdf - Bio-Nica.info

Recombinant PCR 521
Fig. 3. Diagram illustrating site-directed mutagenesis of a single site using recombination
PCR with 4 primers. The primers are numbered hemiarrows. The asterisk designates the
mutagenesis site. Primer 2 is complementary to primer 4. Restriction endonuclease sites A and
B bracket the insert. Notches designate point mismatches in the primers and resulting mutations
in the PCR products. There is no purification of the PCR products. For each additional single
site-directed mutagenesis reaction, only a new primer 1 and 3 need to be synthesized, and the
same cut templates can be used. Reprinted by permission from Technique 2, 273–278.
a. Use 50 µL of E. coli for each sample transformed, because this is effective and less
expensive than the 100 µL recommended.
b. After incubation at 37°C in a shaker for 1 h, plate the entire sample onto an LB plate
containing 100 µg/mL ampicillin.
c. Once an aliquot of bacteria is thawed, do not use it again.
Then, set up the following transformations: Plate A: 2.5 µL of PCR 1 + 2.5 µL of PCR 2;
Plate B: 2.5 µL of PCR 1 + 2.5 µL of TE; Plate C: 2.5 µL of PCR 2 + 2.5 µL of TE; Plate
D: 0.5 ng of a supercoiled template in 5 µL of TE; and Plate E: 5 µL of TE.